Inbred miniature swine provide a unique preclinical model for the study of transplantation immunity and tolerance. Over the past 18 years, we have utilized this model to study a robust form of tolerance of MHC class I- mismatched renal allografts that is routinely achieved following a short course of calcineurin inhibitors. This proposal represents a G37 renewal of the previous long-standing R01, which received a Merit Award at the time of the last competitive renewal. During the past three and one-half years since that renewal, we have made significant progress in understanding the role of regulatory T cells (T-reg) in determining the balance between alloreactivity and down-regulation of immune reactivity to tolerated renal allografts. Among these studies, has been an evaluation of the possible role of antigen presentation through direct or indirect pathways of activation in the reinforcing of tolerance by donor-type skin allografts in previously tolerant animals from which the tolerizing graft has been removed. We have obtained considerable evidence for dominance of down-regulation by the direct pathway in this phenomenon. In the next project period, we intend to: 1) Complete our studies on the nature of cell populations responsible for adoptive transfer of tolerance that are still in progress; 2) Determine whether the thymus is required for breaking and/or reinforcing tolerance after graftectomy in animals tolerant of class I mismatched renal allografts; and 3) Examine the mechanism by which B cell immunity to class I antigens is controlled in animals tolerant of a class I-mismatched renal allograft through evaluation of the influence of tolerance on antibody fine specificity. The broader goal of these studies remains to develop an understanding of the mechanisms by which allograft tolerance is induced and maintained in this large-animal model, in order to permit development of appropriate protocols for induction of tolerance to organ allografts in the clinic.